Generating Double-Crossover Recombinants of Pseudomonas aeruginosa for Targeted Gene Deletion

Abstract

Source: Valentine, M. E., et al. Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection. J. Vis. Exp. (2020).

This video demonstrates the generation of double-crossover recombinants in Pseudomonas aeruginosa using a plasmid-based gene deletion strategy. The process involves selecting bacteria that harbor a non-replicative plasmid integrated into the genome via a single crossover and then inducing a second recombination event under sucrose selection to excise the plasmid. Successful double-crossover recombination is confirmed by the loss of antibiotic resistance and the ability to grow on sucrose-containing media.

Protocol

1. Detection of Double-crossover Recombinants of P. aeruginosa

1. For each broth culture, inoculate 10 µL of culture onto a pre-warmed plate of PIA (Pseudomonas Isolation Agar) supplemented with 10% sucrose (without glycerol) and streak for isolated colonies. Incubate plates overnight at 37 °C.
2. The next day, remove plates from the incubator and inspect them for growth. Sucrose-resistant colonies should be double-crossover recombinants (i.e., have removed the plasmid from the chromosome via a recombination event between the other homologous region of the plasmid insert and the P. aeruginosa chromosome).
   1. Patch at least 20 colonies with sterile toothpicks onto pre-warmed plates of: 1) PIA, 2) PIA supplemented with 10% sucrose (without glycerol), and 3) PIA supplemented with 300 µg/mL of carbenicillin.
3. Incubate plates overnight at 37 °C.
4. Remove plates from the incubator and examine them for growth. True double-crossover recombinants will be carbenicillin-sensitive and sucrose-resistant (i.e., colonies that grew on PIA and PIA supplemented with sucrose, but did not grow on PIA supplemented with carbenicillin).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Kanamycin monosulfate	Fisher Scientific	BP906-5
LE agarose	Genemate	3120-500	via Fisher Scientific
Luria Broth	Difco	240230	via Fisher Scientific
MicroPulser Electroporator	BioRad	1652100
Noble agar, ultrapure	Affymetris/USB	AAJ10907A1	via Fisher Scientific
Phosphate buffered saline powder	Sigma	P3813-10PAK	Sigma-Aldrich
Pseudomonas isolation agar	Difco	292710	via Fisher Scientific
Pseudomonas isolation broth	Alpha Biosciences	P16-115	Custom made batch
Shaking Incubator	New Brunswick Scientific	Innova 4080	shake at 200 rpm
Small Plates (100 O.D. x 10 mm)	Fisher Scientific	FB0875713
SmartSpec Plus Spectrophotometer	Bio-Rad	170-2525	or preferred method/vendor
Sucrose	Fisher Scientific	S5-500